Biomaterials, defined as substances other than food or drugs contained in therapeutic or diagnostic systems that are in contact with tissue or biological fluids, play a crucial role as implants, diagnostics, devices for controlled delivery, and extracorporeal devices to name a few (see, Peppas, N. A.; Langer, R., Science, 1994, 263, 1715). One of the most significant problems for the use of devices fabricated from traditional biomaterials is the difficulty in engineering these surfaces to mediate interactions between cells and the biomaterials. The ability to design specific biomaterials, however, necessitates information about the molecular nature of surfaces of cells. Fortunately, advances in our knowledge of biochemical interactions at cell surfaces have led to structure elucidation of ligand molecules that bind to cell surface receptors and influence cell behavior (see, Yamada, K. M. J. Biol. Chem. 1991, 266, 12809; Koivunen et al., Biotechnology, 1995, 13, 165; Pasqualini et al., Nature, 1996, 364). Obtaining this information about the molecular nature of the surface of cells has enabled the development of novel materials for in vitro and in vivo biotechnology applications including the ligands that bind to cell surface receptors. (Hoffman, A. S., Artificial Organs, 1992, 16,43; Peppas, N. A. and Langer, R. S., Science, 1994, 263, 1715; Han, D. K. and Hubbell, J. A., Macromolecules, 1996, 29, 5233; Schaffer et al., Tissue Eng., 1997, 3, 53). Currently, the vast majority of ligand coupling to materials is through covalent attachment of ligands to the bulk polymer. However, attachment to the bulk is often inefficient when the end goal is surface derivatization. For example, many existing biodegradable polymers lack reactive groups that can be used to couple biological molecules to fabricated surfaces (Desai, N. P. and Hubbell, J. A., Biomaterials, 1991, 12, 144; Barrera et al., J. Am. Chem. Soc., 1993, 115, 11010). Even if such reactive groups are available, covalent strategies cannot be applied in all circumstances due to harsh reaction conditions and inefficient coupling or purification methods. In addition, reactive surface functionalities may be masked by surfactants used during system fabrication. Finally, very few techniques are universally applicable to any class of ligand (e.g., peptide, oligosaccharide, lipid) and do not damage the material or the ligand.
Clearly, there remains a need to improve the interactions between cells and biomaterial architectures. It would be most desirable to develop a system in which virtually any class of ligand could be efficiently coupled to a wide range of biomaterial architectures.
The present invention provides a novel surface engineering strategy that uses biomolecular interactions to immobilize surface modifying ligands on biomaterial architectures. The surface modified compositions resulting from the inventive method are useful in many contexts, including, but not limited to, scaffolds for tissue engineering and as vehicles for site specific drug delivery. In general, in a preferred embodiment, this biomolecular interaction is achieved by using an xe2x80x9canchor-adapter-tagxe2x80x9d system, in which an adapter which can interact specifically and with high selectivity with an anchor molecule (present on the biodegradable surface) and a tag (bound to the ligand to be immobilized) simultaneously is used in attaching the ligand to the surface in a manner which is stable in vitro or in vivo. This has the advantage that the ligands may be presented in an active conformation, may be attached to the surface in an aqueous environment, and may be attached rapidly to the surface. In a particularly preferred embodiment, a polymeric material is utilized as the biomaterial architecture, and the anchor and tag comprise biotin, and the adapter comprises avidin or streptavidin.
The method of generating these novel surface modified biomaterials using an xe2x80x9canchor-adapter-tagxe2x80x9d system involves (1) providing a biomaterial architecture, having an anchor attached thereto or incorporated therein; (2) contacting said biomaterial-anchor moiety with an adapter moiety; and (3) contacting said biomaterial-anchor-adapter moiety with a desired ligand having a tag incorporated therein to produce a biomaterial-anchor-adapter-tag-ligand moiety. In certain preferred embodiments, these novel compositions can be utilized in tissue engineering applications and also for applications in site specific drug delivery.
It will be appreciated that a two-component xe2x80x9canchor-tagxe2x80x9d system can also be utilized in the present invention instead of the three-component xe2x80x9canchor-adapter-tagxe2x80x9d system. Thus, in one preferred embodiment, the same moiety can be utilized for the anchor and the adapter, thus providing an xe2x80x9canchor-tagxe2x80x9d system. In another preferred embodiment, the same moiety can be utilized for the adapter and tag, thus providing an alternate xe2x80x9canchor-tagxe2x80x9d system.
The method of generating these novel surface modified biomaterials using an xe2x80x9canchor-tagxe2x80x9d system involves (1) providing a biomaterial architecture, having an anchor attached thereto or incorporated therein; and (2) contacting said biomaterial-anchor moiety with a desired ligand having a tag incorporated therein to product a biomaterial-anchor-tag-ligand moiety.
In another aspect, the present invention provides a method for synthesizing a block co-polymer material having an anchor associated therewith. This method involves (1) providing a biodegradable polymeric material, wherein said polymeric material is capable of having an anchor moiety associated therewith and wherein said polymeric material has at least one functionality capable of further polymerization; (2) contacting said solution with an anchor moiety capable of associating with said polymeric material; and (3) subjecting said polymeric material having an anchor associated therewith to conditions capable of effecting further polymerization at a desired functionality to yield a desired anchor adapted block co-polymer material. This material can then be subsequently adapted into a variety of biomaterial architectures for use in the present invention as described above.
In another aspect, the present invention provides methods for site specific delivery of therapeutic agents comprising (1) providing a composition comprising a biomaterial architecture, wherein said architecture has a biological ligand attached thereto by a biomolecular interaction, and a therapeutic agent associated with the biomaterial architecture; and (2) contacting said composition with cells, wherein the ligand attached to the biomaterial architecture interacts with the cells to effect site specific delivery of the therapeutic agent to the cells.
In yet another aspect, the present invention provides methods for tissue engineering comprising (1) providing a scaffold having a biological ligand attached thereto, wherein said ligand is attached by a biomolecular interaction; (2) contacting said scaffold with cells, wherein said cells interact specifically with the ligand attached to the scaffold; (3) promoting cell growth and/or differentiation to generate tissue; and (4) implanting the tissue.
xe2x80x9cHigh affinityxe2x80x9d: As used herein, the term xe2x80x9chigh affinityxe2x80x9d refers to an interaction with a Kd of between 10xe2x88x9213 and 10xe2x88x926 M.
xe2x80x9cInteracts specificallyxe2x80x9d: As used herein, the term xe2x80x9cinteracts specificallyxe2x80x9d means that the component interacts with at least 100-fold higher affinity (and preferably at least 500-fold, or at least 1000-fold, or at least 2000-fold higher affinity) with the intended binding component than with other molecules that may be encountered by either of the said components when administered to a patient.
FIG. 1 depicts a particularly preferred embodiment of the present invention.
FIG. 2 depicts a comparison of avidin immunobilization on surfaces of PLA-PEG-biotin and PLA-PEG by surface plasmon resonance analysis.
FIG. 3 depicts a fluorescence confocal micrograph of biodegradable microparticles.
FIG. 4 depicts confocal micrographs of PLA-PEG-biotin microparticles following incubation with avidin-Texas Red and biotin-FITC.
FIG. 5 depicts representative micrographs and cell area histograms of bovine aortic endothelial cells on the PLA-PEG-biotin surface and the RGD-functionalized surface 5 h after cell seeding.